Diversity, seasonal abundance, and environmental drivers of chaetognath populations in North Inlet Estuary, South Carolina, USA

Abstract Chaetognaths (Phylum: Chaetognatha) are one of the most abundant phyla of zooplankton worldwide and play an important role in marine trophic interactions. Although the role of chaetognaths in global ecosystems is well understood, the spatial variation and environmental drivers of estuarine chaetognath populations is poorly understood. To provide the first known record of chaetognath species composition in a coastal estuary in the south‐eastern USA, chaetognaths were identified and quantified from zooplankton samples collected on a monthly basis in 2019 and 2020 from North Inlet Estuary in South Carolina. Parasagitta tenuis was the most abundant species of the five found, making up 33% of total abundance. The egg presence of these chaetognaths was further analyzed to gauge reproductive cycles. Abundance and egg presence were compared with surface and bottom measurements of temperature, salinity, and dissolved oxygen levels to determine the driving abiotic factors behind chaetognath's seasonal variability and reproductive cycles. Temperature, salinity, and dissolved oxygen all had low (r < ±.29), non‐significant correlations with abundance. Chaetognath egg production was most significantly associated with dissolved oxygen (p < .001) and seasonal changes in temperature (p < .001). Our initial findings indicate the continued abundance of chaetognath in a local estuary are dependent on abiotic factors that are strongly influenced by a changing climate.

high abundance, chaetognaths play a significant role in transferring energy to higher trophic levels within marine food webs, as they prey on other mesozooplankton, macroplankton, and small vertebrates such as larval fish (Lebour, 1923;Alvariño, 1975;Kuhlmann, 1977;Baier & Purcell, 1997;Coston-Clements et al., 2009). Several studies have suggested chaetognaths can cause significant mortality in fish larvae, due to their high abundances and the documented presence of larval fish in their guts (reviewed in Alvariño, 1985;Baier & Purcell, 1997).
Chaetognaths reproduce sexually and are hermaphroditic, with individuals possessing both a pair of seminal vesicles and receptacles (Ghirardelli, 1969;Goto & Yoshida, 1985;Pierrot-Bults, 1976). Upon maturation of fertilized eggs, the eggs are expelled from the individual, where they attach to vegetation or develop within marsupial sacs (Terazaki & Miller, 1982). Chaetognaths surpass the larval stage and emerge from their eggs as juveniles. This development occurs quickly, with some species developing from fertilization to juvenile within several days (Goto & Yoshida, 1997;Margulis and Chapman, 2010). Alvariño (1990) suggested chaetognath egg development and seasonal laying times may be dependent on temperature, with laying occurring earlier in the day on warm days and most species breeding primarily in spring, with breeding occurring year-round in tropical waters.
While research into the ecology of chaetognaths, specifically related to their seasonality (Gilmartin et al., 2020;Lozano-Cobo et al., 2017;Nogueira Júnior et al., 2019;Tse et al., 2008), has increased over the last few decades, there is still a gap in our scientific understanding of estuarine species' geographic distribution and the environmental factors which drive their abundance and reproduction. O'Brien (1977) and Tiselius and Peterson (1986) suggested chaetognath distribution and species composition depend on temperature and salinity levels. Both Lonsdale and Coull (1977) and Houser and Allen (1996) verified the presence of chaetognaths in North Inlet Estuary, but did not identify them to species level. Johnson and Allen (2012) provided details on the behavior, morphology, and range of zooplankton species within the coastal zone of the southeastern USA, which included six species of chaetognath (Parasagitta elegans (Verrill, 1873; formerly known as Sagitta elegans), Parasagitta tenuis (Conant, 1896; formerly known as Sagitta tenuis), Flaccisagitta enflata (Grassi, 1881; formerly known as Sagitta enflata), Ferosagitta hispida (Conant, 1895; formerly known as Sagitta hispida), Sagitta helenae, and Sagitta bipunctata). Parasagitta tenuis, Parasagitta elegans, Sagitta helenae, and Ferosagitta hispida can primarily be found in neritic waters, whereas S. bipunctata is a predominantly oceanic species (Avila & Cadena, 1999;Owre, 1960;Thuesen et al., 1993;Tovar et al., 2009). Previous research on chaetognaths in the southeastern USA has focused on determining species composition and the reproductive seasonality of chaetognaths (Owre, 1960;Pierce & Wass, 1962), as well as the interaction between chaetognaths and larval fish (Coston-Clements et al., 2009).
As chaetognath abundance in North Inlet Estuary has been previously shown to have a positive relationship with temperature (Houser & Allen, 1996), we hypothesize temperature to act as a tracer for abundance and that chaetognath abundance and egg presence will demonstrate similar seasonality following the climate of the estuary. There are several other factors likely impacting chaetognath seasonality, including prey availability and predator abundance. We hypothesize the presence and fluctuating abundances of chaetognath in North Inlet Estuary has the potential to alter copepod stock.
While this study does not aim to assess actualized impacts of chaetognaths on copepod stocks in North Inlet Estuary, through the use of ranges of chaetognath digestion rates and average numbers of prey per chaetognath found in previous literature, as well as previous data on copepod stock within the estuary, and chaetognath abundances found in this study, we can make estimates regarding copepod predation in the system.
The purpose of this study is to provide the first known record of chaetognath species composition within the North Inlet-Winyah Bay estuary, while contributing a local dataset regarding their reproductive cycle and its driving forces. Understanding the seasonality of chaetognath abundance and egg presence in the North Inlet Estuary can aid in our understanding of marine food webs, as well as chaetognath's seasonal effects on the copepod population in the system. F I G U R E 1 Preserved and stained Parasagitta tenuis collected from North Inlet Estuary.

| Description of study area
The study was conducted in North Inlet Estuary, in Georgetown County, South Carolina (Figure 1), a barrier-island-bounded system with temperate, high salinity water that is primarily surrounded by a Spartina alterniflora marsh (Allen et al., 2008). Approximately 90% of the estuary's watershed is protected by a private foundation and has remained undeveloped over the past 200 years, keeping the estuary relatively free from local anthropogenic impacts (Allen et al., 2014).
The sampling site is roughly 2 km from the ocean and is at the confluence of Town, Bread and Butter, and Clambank creeks (79.1877° W, 33.3320° N). The depth in the western portion of the channel where sampling occurred is ~2 m at low tide, with an average semidiurnal tidal range of 1-2 m. The bottom is a sand-mud matrix and due to the shallow water depth and high tidal currents, the creek is generally well mixed and vertically homogenous with respect to dissolved substances. Additionally, due to the high input of oceanic waters and protected watershed, in the past nutrient concentrations in the estuary were low and water quality was high (Allen et al., 2008;Dame et al., 1986).
The salinity of North Inlet Estuary is primarily controlled by the semidiurnal exchange of water with the coastal ocean, with small amounts of freshwater input from a forested watershed. Salinity in North Inlet usually remains high (30-35) except when there are influxes from heavy local rainfall, which can reduce salinity concentrations to below 20 for several days. Similarly, when the brackish water from Winyah Bay to the south moves into the North Inlet system, salinity levels can be reduced significantly (15-25) for a time span of weeks to months (Allen et al., 2008;Traynum & Styles, 2007).
Estuarine systems are highly dynamic with respect to their hydrology, nutrient cycling, and biotic resource management. Estuaries' complex mixture of tidal saltwater and freshwater runoff are shaped by certain climatic forcing features, such as temperature, amount of rainfall, and wind patterns (Paerl et al., 2010). The North Inlet is a system vulnerable to the extreme effects of these long-term changes to the natural system and has shown an increase in average temperatures of 1.5°C over the past 40 years (Allen et al., 2014) (Figure 2).

| Sampling design
The sampling design followed the methods of Houser and Allen (1996). Zooplankton samples were collected during one tow between 1000 and 14,000 h each month during outgoing mid-tide from November 2019 to November 2020. Tows were carried out horizontally, 20 m from the marsh bank, parallel to the shore, and in the direction of the ebbing tide. Each tow lasted roughly 5 min and covered 200 m. The tows were conducted aboard a boat with an epibenthic sled equipped with a 365μm mesh net, a mouth opening of 50 cm wide by 35 cm high, and with a general oceanics flowmeter attached to the net's mouth. During the tow, the net mouth was just above the bottom, ~1.5 m below the surface water. Surface (~0.5 m below surface water) and bottom (~1.5 m below surface water) salinity, temperature, and dissolved oxygen were measured from an anchored boat using a YSI Pro 2030 Sonde.
Upon collection, samples were immediately preserved in 4% borax-buffered formaldehyde-seawater solution. Samples were then processed in the lab using a dissecting microscope at 10-40× magnification. For samples with high volumes of animals and small detritus, a Folsom splitter was used to subsample. All chaetognaths found were identified to species using Johnson and Allen (2012), and subsequently counted and classified by egg presence. Egg presence was quantified by the visual presence of eggs. If egg presence was visible, then the chaetognath was counted as having eggs present, whereas if no eggs were visible, the chaetognath was classified as having no eggs present.

| Data analysis and statistics
Abundance values for the number of individuals with eggs present, no eggs present, and total chaetognaths were converted into the volume of animals per m 3 using the 0.175 m 2 mouth opening and flowmeter readings for each sample. Unidentifiable fragments of chaetognaths found were labeled as "Unknown Chaetognath" (UK) and included in statistical analyses of total chaetognath abundance.
Two samples were analyzed in November and March, respectively; the chaetognath abundance and accompanying environmental data were averaged for the two samples within each month.
All statistical analyses were done using R Statistical Software To estimate chaetognath's impact on copepod stock in North Inlet Estuary, we used the formula derived by Bajkov (1935) as seen in Feigenbaum & Maris (1984) and Baier & Purcell (1997): where FR N is the daily feeding rate (prey per chaetognath per day), NPC is the prey per chaetognath, T is the time period in hours, and DT is digestion time in hours. T = 24 was used for all calculations. As consumption and digestion times can vary between species (Feigenbaum & Maris, 1984;Baier & Purcell, 1997), we utilized average values for digestion times and prey per chaetognath values for only S. helenae and F. hispida as these were the only species found in both this study and Baier and Purcell (1997). Digestion time was calculated by observing isolated chaetognaths with prey and recording the times (in 15 min intervals) of initial isolation, prey time, and position within the gut (Baier & Purcell, 1997). Chaetognaths were observed until the prey was no longer visible in the gut, then digestion time estimates were multiplied by two to account for digestion that may have been occurring prior to observation (Baier & Purcell, 1997

| Abundance and egg presence
Chaetognath abundance averaged across all samples was 3.  Higher abundances and more species were found at lower dissolved oxygen levels (Figure 7).    (Lonsdale & Coull, 1977) and

RDA analysis indicated some seasonality in species composition
and abundance, both of which varied mostly with surface temperature axis (Figure 8). Houser and Allen (1996) Alvariño, 1990), with laying occurring earlier in the day on warm days and most species breeding primarily in spring, although noting breeding does occur year-round in tropical waters.
Spawning times appear to vary by species, with P. elegans and P. tenuis seemingly spawning year-round. Zo (1973) found P. elegans had two seasonal egg development periods, spring and autumn, where high abundances of eggs were present. The highest abundances of eggs present in Bedford Basin, Nova Scotia, occurred from March to June, as well as September to December (Zo, 1973). Similarly, this study found P. elegans highest period of egg presence occurred in spring and summer (April, May, and July) as well as fall (October and November). Owre (1960) found S. bipunctata did not have yearround spawning times, mainly reproducing in the early spring, summer, or in late winter, and S. helenae and F. hispida did reproduce year-round, with S. helenae having an accelerated breeding rate during May and June, while F. hispida had the highest reproduction rates in mid-winter and spring. These findings support the data collected in this study, which found F. hispida to have the highest egg presence rates occurring in spring (April) and S. helenae's egg presence peaking in summer (July). There were not enough individuals found in this study to make conclusions regarding S. bipunctata's reproductive seasonality.  et al., 2019;Owre, 1960;Pierce, 1951;Pierce & Wass, 1962;Ulloa et al., 2000). While this study found weak correlations between salinity and chaetognath abundance, primarily oceanic species (such as F. enflata, S. bipunctata, and Sagitta megalopthalma) have demonstrated significant positive relationships between chaetognath assemblages and salinity levels (Gilmartin et al., 2020). This likely indicates the species found in this study have higher tolerances to wide salinity fluctuations, which would account for the very low abundance of S. bipunctata observed in North Inlet Estuary. Pierce and Wass (1962) found P. tenuis and S. helenae demonstrated a wide temperature and salinity tolerance, whereas F. hispida demonstrated salinity tolerance up to 36.
Similarly, Pierce and Wass (1962) found S. bipunctata was more Chaetognaths play an important role in marine trophic interactions and understanding their seasonality is important for our understanding of copepod populations. The impact of chaetognaths on copepod populations was estimated by Baier and Purcell (1997), who found that chaetognaths can consume up to 44% of large copepod standing stocks per day over the mid-shelf area of the southeastern USA. Average abundances of copepods in the studied region of North Inlet Estuary are ~7000 copepods m −3 (Stone and Allen, unpublished data), meaning chaetognaths in this study on average consume 0.010% of copepod standing stock each day, and at their most abundant, could consume 0.034%. As this number does not account for variations in chaetognath species composition, it is important to note that our estimations include an element of error, as consumption and digestion rates can vary between species (Feigenbaum & Maris, 1984). While chaetognaths in some systems can exert large top-down pressure on the copepod community (Tönnesson and Tiselius, 2005), based on our calculated feeding rates and abundances, we found no evidence for high predation pressure, on average, in this system. However, as total abundances of chaetognaths in North Inlet Estuary has been reported much higher than those ob- While the study site has experienced long-term warming, specifically in winter months, salinity in the estuary has been variable but has not changed significantly in the long term, indicating relatively stable levels of freshwater run-off in the system (Allen et al., 2014).
However, short term fluctuations in salinity due to increased run-off likely did affect chaetognath assemblages in the system. Further, diel vertical migration, in which organisms migrate vertically through the water column for prey, protection, or resource partitioning, has been shown to impact chaetognath abundance (Sweatt and Forward, 1985).
However, diel vertical migration likely played little role in the observed chaetognath abundances in this study, as chaetognath abundance in North Inlet Estuary is greatest during the daytime and not related to depth (Houser and Allen, 1996).
A potential cause for the relatively low levels of chaetognath abundance found in this study could lie within the sampling design.
While this study included one sampling location within an estuary at a shallow depth (~2 m), similar studies in the region sampled at a variety of different depth locations, with some stations located roughly 200 miles from shore (Coston-Clements et al., 2009;Pierce & Wass, 1962). These differences in sampling designs are likely major sources of uncertainty in this study and may convolute comparisons between this study and research in similar regions.
The low abundance seen in this study, however, may still be an accurate representation of chaetognath presence in the estuary.  et al., 2008). Additionally, during this period there was an observed 26% decrease in chaetognath abundance compared to the longterm mean (Allen et al., 2008). This could indicate that the low numbers found in this study is representative of the estuary, and while the estuary experiences occasional large spikes in abundance (>150 ind. m −3 observed in Allen et al., 2008), chaetognath abundances are typically low.

ACK N OWLED G M ENTS
Thanks to the many technicians, volunteers, and researchers who were part of the North Inlet Estuary Zooplankton Time Series during the study period, especially Bruce Pfirrmann and Dennis Allen.
This project was supported by the North Inlet-Winyah Bay National Estuarine Research Reserve.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available in Dryad at doi: 10.5061/dryad.4qrfj6qft.